铜绿假单胞菌LexA蛋白在大肠杆菌中的表达与纯化

目的克隆铜绿假单胞菌PAO1株的LexA蛋白编码区，在大肠杆菌中表达并纯化表达蛋白。方法提取铜绿假单胞菌PAO1株基因组DNA，PCR扩增LexA蛋白编码区，A—T克隆于质粒pMD18-T载体中。阳性重组子经酶切后，回收目的片段连接至相应线性化的表达型载体pET-28a（＋）中，重组质粒经测序鉴定后，转化大肠杆菌BL21感受态，筛选高效表达株，表达产物经SDS-PAGE初步鉴定后，用镍珠吸附一步法纯化，表达蛋白转印PVDF膜后，经N-端测序证实。结果本研究成功克隆到PAO1株的LexA蛋白编码区并在大肠杆菌中获得表达，纯化表达蛋白经N-端测序证实确为PAO1的LexA蛋白。结论研究结果为铜绿假单胞菌PAO1株基因组中SOS盒的实验鉴定及LexA蛋白调控基因的筛选奠定了基础。

Expression and purification of the LexA protein in Pseudomonas aeruginosa in E. coli

The genomic DNA was extracted from Pseudomonas aeruginosa strain PAO1,and the coding sequence of LexA protein was amplified and linked to vector pMD-18T.The recombinant plasmid was digested with NcoI and XhoI;and the fragment with size of 650 bps was recovered and subcloned into the expression vector pET-28a(+).Then,the recombinant plasmid was transformed to E.coli BL21.After being sequenced,the expressed product was identified with SDS-PAGE by one step method of Ni-pearls absorption,and this protein was transferred to PVDF membrane then sequencing at N-terminus by Edman's degradation method.The experimental results showed that the LexA protein of P.aeruginosa could be expressed in E.coli,and after purification,the N-terminus sequencing analysis proved that the expressed product was the LexA protein of PAO1 and its purification rate could reach up to 96%.It is concluded that this work provides a basis for the identification of SOS box and LexA regulated gene in the genome of P.aeruginosa strain PAO1.